A research program is described to study the role of uncoupling protein-2 (UCP2) in the pathogenesis of steatohepatitis (SH). SH evolves from fatty liver, a prevalent condition among patients with obesity, diabetes, and alcohol abuse. The predictors and biochemical mechanisms of this progression are poorly defined, but reactive oxygen species (ROS) and endotoxin-mediated cytokine release appear to have a major role. UCP2 is a mitochondrial inner membrane protein emerging as a potential regulator of mitochondrial ROS production. Expression of liver UCP2 is markedly increased in animal models of obesity, alcohol intake, and endotoxin exposure. We hypothesize that induction of UCP2 in the liver is a defensive mechanism and it may involve both parenchymal hepatocytes and Kupffer cells. UCP2-/- mice generated in the mentor's laboratory will provide a powerful tool to test this hypothesis. First, steatosis will be induced in UCP2-/-mice by crossbreeding with ob/obmice, high-fat feeding, and ethanol feeding. UCP2-/- mice will also be challenged by in vivo treatment with endotoxin, TNF-alpha, and anti-Fas antibody. UCP2-/- mice are expected to show accelerated liver injury under these conditions. Adenovirus-mediated gene transfer will be used to rescue UCP2 functions in vivo. The cellular mechanisms of UCP2 action will then be studied in vitro in cultured parenchymal hepatocytes and Kupffer cells. Apoptosis and ROS production will be detected in hepatocytes from UCP2-/- and wild type mice by fatty acids, ethanol, and ceramide. SOD mimetics and scavenger compounds will be used to revert the action of absent UCP2. The effect of UCP2 on various steps of apoptosis will be investigated (mitochondrial permeability transition opening, Bid translocation): The ability of Kupffer cells to generate ROS and release cytokines in the absence of UCP2 will be assessed. Finally, Cre/lox recombinase system will be used to create tissue-specific UCP2 knockout mice. Mice with a loxP-modified UCP2 allele will be created and bred with mice expressing an albumin promoter-driven Cre to obtain hepatocyte-specific UCP2 knockouts. Breeding of UCP2/loxP mice with mice expressing a lysozyme promoter-driven Cre will result in Kupffer cell/macrophage-specific UCP2 knockouts. Generation of Cre/lox system for tissue-specific UCP2 expression will provide a great learning potential for the applicant. The research facilities of the mentor's laboratory will provide an excellent environment to accomplish these aims. Knowledge of the biochemical actions and genetic regulation of UCP2 in the liver will advance our understanding about the role of UCP2 and the pathophysiology of SH.